Investigations on the regulation of normal intestinal immunity have described two fundamental properties of the intestinal immune system that are distinct from other peripheral and tissue responses. The first is "oral tolerance," in which dietary antigen leads to systemic non-responsiveness. The second is "physiological inflammation," a state of immune regulation in the intestine characterized by hypo-responsive T lymphocytes. These unique processes indicate that active tolerance is an essential component of mucosal immunity and we propose that regulated mucosal T cell hypo-responsiveness mediates intestinal immune tolerance. In striking contrast to circulating T cell, which are activated via the CD3 pathway, intestinal lamina propria T cell (LPT) responses are dominated by an alternate pathway that uses the CD2 receptor. We have developed an in vitro model in which LPT can toggle between a hypo-responsive (tolerant) state and a responsive (protective) state of activation. Using this model we demonstrated that membrane proximal signaling within 2 minutes of activation through the CD3 receptor complex is markedly reduced in tolerant, but not protective LPT. Recent published work in mesenchymal cells and our submitted manuscript studying PBT have described the function of cholesterol-rich membrane microdomains, called lipid rafts, as not only structural components of the plasma membrane, but also localized sites for the initiation of intra-cellular signal transduction. With our expertise in characterizing LPT, isolating lipid rafts, and delineating signal transduction pathways in isolated T cells, we are immediately poised to study mucosal T cell tolerance at a molecular level. Our ability to identify and characterize the heterogeneity of lipid raft constituents in LPT will be combined with molecular, biochemical, and immunological techniques to test the following central hypothesis: Mucosal T cell tolerance is maintained by the differential targeting to discrete lipid rafts of proteins that regulate T cell activation via the CD3 and CD2 receptors. Our aims are as follows: (1) Determine the association of negative regulators of TCR signaling with lipid rafts in tolerant LPT. (2) Investigate the coupling of the cytoskeleton to lipid rafts via interactions through CD2 in LPT. (3) Investigate how the second messenger Ras-MAPK pathway is regulated by lipid raft associations in LPT. We believe this unique approach will generate important new information on intestinal immune tolerance by examining the regulation of membrane proximal events in LPT signaling and how these modulate mucosal T cell mediated immunity.